We are losing the battle against antibiotic-resistant Gram-negative bacterial pathogens in our hospitals and clinical care facilities. The CDC estimates that over 2 million people are infected annually with 17 species of antibiotic-resistant bacterial pathogens, killing 23,000 people per year. Over half of the species are Gram- negative pathogens including carbapenem-resistant Enterobacteriaceae (CRE) and the non-fermenting opportunistic pathogen Acinetobacter baumannii. Although the CDC described carbapenem-resistant (CR) bacteria as ?nightmare bacteria?, the factors required for virulence of these pathogens or their antibiotic- susceptible counterparts during bloodstream infections are largely unknown. Thus, there is an urgent need to identify unique (species-specific) and common (required by most of the six Gram-negative species) fitness and virulence factors required by these species for bacteremia, and map key metabolic pathways and essential gene sets used by these pathogens in vivo. Our long-term goal is to delineate the mechanisms of pathogenesis in Gram-negative bacteria that cause hospital-acquired infections. The overall objective of this application is to conduct RNA-seq and measure in vivo growth rates in representative isolates of E. coli, Klebsiella pneumoniae, Serratia marcescens, Citrobacter freundii, and Enterobacter cloacae as well as A. baumannii, in the murine model of bacteremia in which Tn-seq has been largely completed for these isolates. Our central hypothesis is that based on the relatedness of CR species at the family (Enterobacteriaceae) and class (A. baumannii) levels, these pathogens require a combination of orthologous core functions and species-specific fitness factors to acquire nutrients and evade host responses during bacteremia. The rationale for these proposed studies is that antibiotic resistance is rising rapidly in Gram-negative pathogens that cause bloodstream infections in our health care systems. We plan to objectively identify unique and common genes critical for bloodstream infection by antibiotic-susceptible and CR bacteria and measure their in vivo gene expression. Unique and common virulence determinants will be investigated to determine mechanisms of pathogenesis. We will test our central hypothesis and attain the objective of this application by completing these specific aims: 1) Define the active metabolic pathways and resultant growth kinetics across six Gram-negative pathogens during bacteremia. 2) Identify shared and unique pathways required for bacteremia by six Gram-negative pathogens. Specific expected outcomes will include: (a) precise measurement of growth kinetics of each pathogen during bacteremia, (b) identification of preferred and required pathways at equivalent growth phases in vivo, and (c) detailed maps of these pathways annotated with genes that are pathogen-specific or shared across multiple pathogens. The positive impact of these studies will be substantial. We will uncover mechanisms of pathogenesis for six of our most concerning antibiotic-resistant hospital pathogens and identify targets for developing new therapeutics.